Device and method for separation of proteins and other biomolecules

ABSTRACT

The present invention relates to a device and method for separation of proteins and other biomolecules. A preferred use is for sample preparation of crude as well as pre-fractionated samples. In a preferred embodiment, the device of the present invention is a pipette tip having dual channels, one for inlet of sample and one for outlet. The outlet, but not the inlet, channel is provided with sample separation media for separation of a desired biomolecule from a sample. The flow through the sample separation media is unidirectional.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. §371 and claims priority tointernational patent application number PCT/SE2008/000341 filed May 20,2008, published on Dec. 4, 2008, as WO 2008/147281, which claimspriority to patent application number 0701316-2 filed in Sweden on May30, 2007.

FIELD OF THE INVENTION

The present invention relates to a device and method for separation ofproteins and other biomolecules. A preferred use is for samplepreparation of crude as well as pre- fractionated samples. In apreferred embodiment, the device of the present invention is a pipettetip having dual channels, one for inlet of sample and one for outlet.The outlet, but not the inlet, channel is provided with sampleseparation media for separation of a desired biomolecule from a sample.

BACKGROUND OF THE INVENTION

It is often desired to separate target molecules from complex mixtures,such as body fluids. The target molecule may be any biomolecule presentin such a fluid, for example protein, peptide, nucleic acid andcarbohydrates. Most often this requires pre-fractionation of the sampleor some kind of sample preparation. Sample preparation is conventionallyperformed by chromatography or electrophoresis.

For chromatography on columns the sample flow, in binding or elutionmode, is in one direction, i.e. the sample is usually loaded on the topof the column and collected at the bottom of the column.

Another way of achieving sample preparation is by using pipette tipsfilled with separation media. In prior art pipette tips for samplepreparation, the sample is drawn into the pipette tip and is allowed toreact with the separation media in the tip. The sample is drawn into thesame channel of the pipette tip as it is later expelled from the pipettetip when the desired contact with the separation media has occurred.Thus, the flow of sample in the pipette tip is in two oppositedirections, first into the tip and then out of the tip. This has anumber of drawbacks, for example the separation media will becontaminated with sample constituents already when the sample is movinginto the pipette which impairs the separation of the desired targetmolecules when the sample is flowing out of the tip and collected.Furthermore, the separation media in the pipette tip may be clogged withsample proteins and other molecules on their way up in the tip whichwill make it impossible to elute any proteins of interest in the outflowfrom the tip.

Therefore, there is a need within the sample preparation area to providea convenient device which avoids the above drawbacks but still providesthe desired separation.

SUMMARY OF THE INVENTION

The present inventors provide a separation device, preferably in theform of a pipette tip comprising a separate inlet and outlet. The deviceprovides for uptake of sample in the inlet which does not come intocontact with sample separation media present in the outlet. The sampleseparation media is only located in the outlet of the device. The sampleis contacted with and eluted from the sample separation media in theoutlet in regular manner. This increases reproducibility and decreasescontamination problems.

In a first aspect, the invention relates to a sample separation devicefor separation of a biological sample, comprising and inlet channel andan outlet channel, wherein only the outlet channel is provided withsample separation media, and wherein the sample is drawn into the inletchannel and only comes in contact with the sample preparation media inthe outlet when the sample is expelled from the device.

Preferably, the sample preparation device comprises a pipette tip withseparated inlet and outlet channels. A purpose of having two separatechannels for inlet and outlet, respectively, is to prevent prematurecontact between the sample constituents and the separation matrix in theoutlet. Another purpose is to enable unidirectional flow in the inletand outlet channel, respectively. Preferably, the inlet and/or theoutlet channels are provided with back valves for securingunidirectional flow.

The pipette tip may be manually or automatically arranged on a pipette.Several pipette tips may be used in a pipette manifold. The proceduremay be performed manually or automatically.

In one embodiment, the inlet and outlet channels are arrangedsubstantially in parallel.

In a further embodiment, the inlet channel is arranged centrically inthe outlet channel.

In yet a further embodiment, the outlet channel is arranged centricallyin the inlet channel.

Only the outlet channel, and not the inlet channel, is provided withseparation media.

Preferably, the inlet and outlet channels are arranged physicallyseparated from each other.

The separation media in the outlet channel may be any separation mediathat separates biomolecules from each other. Examples of separationmedia are size exclusion media, ion exchange media, IMAC (immobilisedmetal affinity chromatography) media, affinity media, HIC (hydrophobicinteraction) media, RPC (reversed phase chromatography) media or anycombination thereof. Other examples of separation media are monoliths,membranes, filters and hydrogels, which may be used in the outlet of adevice according to the invention and which requires unidirectional flowof sample.

The device according to the invention may be used for any type ofseparation with the above media. It may be separation of samplecomponents from other sample constituents, such as other biomolecules orcellular components. Another option is to use the device for moregeneral purposes, such as desalting a sample. In this case theseparation media may be a size exclusion media or a filter.

Preferably, the inlet channel is provided with a back valve at the upperend thereof. The back-valve function of the inlet for one-way flow canbe constructed in different ways but the main purpose is to prevent thesample from flowing out of the device in the same channel as it enteredthe device. In some embodiments, also the outlet channels may beprovided with a back valve as will be described more closely below.

Several embodiments are possible, for example a) double pipette tipswherein you soak in one tip (i.e the inlet channel) and elute throughthe other tip (i.e. outlet channel) which contains the matrix b) acapillary as inlet channel in the middle of a wider outlet channel; andc) a monolith which is not bond to the wall which allows the solution topass through on the outside thereof.

In yet a further embodiment, the inlet channel is physically arrangedoutside the pipette tip, for example as a thin tube adhered to theoutside of a pipette tip which will be provided with separation mediaand serve as outlet channel.

Preferably, the inlet channel is narrower than the outlet channel toprovide more space for the outlet channel in which the samplepreparation is performed.

The sample preparation media is selected from chromatography beads,hydrogels, monoliths, membranes, filters or a combination thereof.

In a second aspect, the invention relates to a method for samplepreparation of a biological sample, comprising the following steps a)drawing sample into an inlet channel of a device as described above; b)contacting the sample with separation media in an outlet channel; and c)expelling sample from the outlet channel, wherein the sample only comesin contact with the sample separation media when the sample is expelledfrom the device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a and FIG. 1 b are schematic views of a pipette tip for samplepreparation according to the present invention.

FIG. 2 a and FIG. 2 b are schematic views of an alternative embodimentof a pipette tip.

FIG. 3 a and FIG. 3 b are schematic views of yet another alternative ofthe pipette tip of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in connection with some non-limitingexamples with referral to the accompanying drawings. It is to beunderstood that the drawings only are examples of the sample preparationdevice of the invention which is only to be limited by the scope of theappended claims.

Examples

Below the present invention will be disclosed by way of examples, whichare intended solely for illustrative purposes and should not beconstrued as limiting the present invention as defined in the appendedclaims. All references mentioned below or elsewhere in the presentapplication are hereby included by reference.

Example 1

This example refers to FIG. 1 a and FIG. 1 b. A pipette tip is shownwith dual tips, 1 and 2. The tip 1 functions as inlet for incoming flowof sample, for example serum (FIG. 1 a). When the sample has been drawninto the upper compartment 3 of the pipette tip, then it is expelledthrough the second tip 2, which functions as an outlet for the sample.The shaded area in outlet 2 represents a separation matrix. To preventsample from enter the second pipette tip with the incoming flow andprevent sample to enter the same way it entered the tip during elution,two back valves are provided one at the lower end 5 of the second tip 2and one at the upper end 4 of inlet 1, which only allows flow indownwards direction as shown by the arrow in FIG. 1 b.

Example 2

This example refers to FIG. 2 a and FIG. 2 b. In this embodiment, aninlet 6 is provided as a capillary centered in the outlet 7. The inlet 6allows entry of incoming flow of sample, for example serum (FIG. 2 a).When the sample has been drawn into the upper compartment 8 of thepipette tip, then it is expelled through the outlet 7, which surroundsthe capillary inlet 6. The shaded area in outlet 7 represents aseparation matrix. To prevent sample from exiting the pipette tip thesame way it entered the tip, a back valve 9 is preferably provided atthe upper end of inlet 6 which only allows flow in downwards directionas shown by the arrows in FIG. 2 b.

Example 3

This example refers to FIG. 3 a and FIG. 3 b. The shaded area representsa separation matrix, in this case a monolith. In this embodiment theincoming flow of sample enters the pipette tip while the monolith whichis not covalently bond stays at the bottom through gravity. The monolithis pushed up with the incoming flow, FIG. 3 a, and the flow passes inthe interspace between the monolith and the pipette walls. When the flowstops the monolith falls back to the bottom of the pipette due to thegravity forces. When the sample has been drawn into the uppercompartment 10 of the pipette tip, then it is expelled through themonolith, FIG. 3 b. The monolith is pushed down by the flow and all thesample has to pass through the monolith. In this case a back valve isnot needed as the only flow possible in a downward direction will bethrough the desired monolith.

It is to be understood that any feature described in relation to any oneembodiment may be used alone, or in combination with other featuresdescribed, and may also be used in combination with one or more featuresof any other of the embodiments, or any combination of any other of theembodiments. Furthermore, equivalents and modifications not describedabove may also be employed without departing from the scope of theinvention, which is defined in the accompanying claims.

1. A sample separation device, comprising an inlet channel (1) and anoutlet channel (2), which outlet channel is provided with sampleseparation media, wherein the sample is drawn into the inlet channel andonly comes in contact with the sample separation media in the outletchannel when the sample is expelled from the device.
 2. The sampleseparation device of claim 1, wherein the device is a pipette tip withseparated inlet and outlet channels.
 3. The sample separation device ofclaim 2, wherein the inlet and outlet channels are arrangedsubstantially in parallel.
 4. The sample separation device of claim 2,wherein the inlet channel is arranged centrically in the outlet channel.5. The sample separation device of claim 2, wherein the outlet channelis arranged centrically in the inlet channel.
 6. The sample separationdevice of claim 1, wherein the inlet channel is provided with a backvalve.
 7. The sample separation device of claim 1, wherein the outletchannel is provided with a back valve.
 8. The sample separation deviceof claim 3, wherein the inlet channel is arranged outside the pipettetip.
 9. The sample separation device of claim 1, wherein the inletchannel is narrower than the outlet channel.
 10. The sample separationdevice of claim 1, wherein the sample preparation media is selected fromchromatography beads, hydrogels, monoliths, membranes, filters or acombination thereof.
 11. A method for sample separation of a biologicalsample, comprising the following steps: a) drawing sample into an inletchannel (1) of the device of claim 1; b) contacting the sample withsample separation media in an outlet channel (2); and c) expellingsample from the outlet channel, wherein the sample only comes in contactwith the sample preparation media when the sample is expelled from thedevice.
 12. The method of claim 11, wherein the sample separation devicecomprises a pipette tip with separated inlet and outlet channels.